The invention relates to a reagent for coagulating blood and for measuring the coagulating activity of a plasma specimen by using optical measuring apparatus or viscosity detecting apparatus, and more particularly to a reagent for coagulating blood and an apparatus capable of measuring specimen of low coagulating activity, while contributing to enhancement of accuracy in ordinary specimens, By preparing them into a composition so that a larger than ever optical changing quantity or viscosity changing quantity may be obtained.
The mechanism of blood coagulation is explained by reference to FIG. 1. The mechanism of blood coagulation usually occurs in two pathways. One route is called the extrinsic coagulation, in which starting from the tissue thromboplastin released from epidermic cell or the like, the coagulated VII factor is activated, and by this activated coagulated VII factor, the coagulated X factor is activated, being followed by activation of the coagulated V factor, II factor, and finally fibrinogen is transformed into fibrin to induce coagulation.
The other route is called the intrinsic coagulation, in which the coagulated XII factor is activated by contact or the like to activate the XI factor. In succession, the activated XI factor activates the IX factor, and further the activated IX factor activates the X factor by collaborating action with calcium ions and VIII factor. It is later followed by activation of the V factor and II factor, and finally fibrinogen is transformed into fibrin to induce coagulation.
Methods for detecting blood coagulation may be roughly classified into the method of increasing viscosity of liquid along with coagulation of blood (viscosity detecting method), the method of detecting white turbidity along with coagulation of blood (turbidity detecting method), and their combined method. In the viscosity detecting method, bar-shaped or spherical magnetic matter is injected into the plasma specimen, and this is mixed with a reagent for detecting coagulation, and the motion of the magnetic matter becomes dull due to coagulation, which is detected. This viscosity detecting method, however, largely differs in the resulting measurements depending on the shape of the fibrin lumps which are final products of blood coagulation (that is, the quantity of fibrin or stiffness of coagulated state), and its fatal defect is that it is impossible to detect unless the viscosity is higher than a specific level. Besides, because of the measuring principle of observing the motion of magnetic matter, the measurement is affected by the intensity of magnetic matter.
The turbidity detecting method measures coagulation only by mixing the plasma specimen and coagulating reagent, and it does not require magnetic matter or other charging. This method of detection is available in the transmitted light detecting method and scattered light detecting method. In these methods of detection, if the fibrinogen quantity is small, the change of the transmitted light quantity or the scattered light quantity can be detected, and it is hence free from the shortcoming known in the viscosity detecting method. However, in either detecting method, it is quite natural that detection is more accurate if the changing quantity of light is greater, and the coagulating reagent to be used desirably has such a characteristic as to express the changing quantity of light very largely.
FIG. 2 is a diagram which explains the change of signal intensity obtained in the scattered light detecting method. The result of investigating the plasma coagulating process by an optical detecting device (scattered light detecting method) is shown. In the diagram, point A is the moment at which mixing of the plasma and coagulating reagent occurs, and afterwards the coagulation reaction progresses in multiple steps, and as a stable fibrin is formed, a change in scattered light appears (point B in the diagram). As the formation of stable fibrin progresses, the change of scattered light increases, but most fibrinogen is consumed, and the change of quantity of scattered light decreases, and the reaction terminates (point C). The coagulating time may be, for example as disclosed in Japanese Laid-open Patent Sho. 59-203959, determined as time T until reaching the 50% scattered light quantity supposing the quantity of scattered light at time B to be 0% and the quantity of scattered light at time C to be 100%. Incidentally .DELTA.H is the changing quantity of scattered light from the start of the coagulation reaction until its end.
To measure the extrinsic coagulation pathway, the method known as Quick's single-stage method is most popular at the present. This measurement (examination) is generally called the PT (prothrombin time) method, which is capable of not only comprehensively measuring the activity of extrinsic coagulating factors, but also measuring individual extrinsic coagulating factors by using coagulating factor deficient plasma. Today, in the patients administered anticoagulants (such as warfarin) for the treatment of heart disease, this measuring method is important as the means for monitoring the effect of anticoagulants.
Most reagents used in this method are prepared by grinding rabbit brain in the presence of acetone, dehydrating, drying and extracting tissue thromboplastin. The tissue thromboplastin differs in reactivity to extrinsic coagulating factors or yield depending on the extraction conditions, and various methods of extraction have so far been invented. For example, Ronald Bach et al. attempted to enhance the yield by solubilizing the tissue thromboplastin by using a surface active agent (Triton X-100) (The J. Biological Chemistry, Vol. 256, No. 16, pp. 8324-8331, 1981). As a similar method, in Japanese Patent Publication Sho. 63-56501, it is proposed to use a surface active agent of the cholic acid group at the time of tissue thromboplastin extraction, and concerning these methods, M. Hvatum and H. Prydz (Biochimica et Biophysica Acta, Vol. 130, pp. 92-101, 1966) and H. Gonmori and Takeda (Thrombos. Haemostas. Vol. 36, pp. 90-130, 1976) reported the method of preparing tissue thromboplastin in detail in their papers. Besides, Japanese Patent Publication Sho. 58-43080 discloses an invention for preparing a useful tissue thromboplastin by maintaining the pH during extraction at the alkaline side.
Whichever method of extraction is selected, it is indispensable to contain tissue thromboplastin in the reagent used in the inspection method (prothrombin time method) known at Quick's single-stage method, and at the same time the tissue thromboplastin must be uniform in the reactivity to extrinsic coagulating factors and must finally possess the action of transforming fibrinogen into fibrin.
For the conventional reagent for coagulating blood (hereinafter called PT reagent), it was the ultimate requirement to have an accurate reactivity to extrinsic coagulating factors and a capability of finally forming fibrin. Little attention was paid to the display of accurate measurement results. Accordingly, significant fluctuations of result of examination about coagulation was allowed.
The problem that the invention is to solve is to prepare such a composition as to enlarge the optical changing quantity in order to display the result of measurements accurately. As a result, not only can a specimen of low coagulating activity be measured, but the invention also contributes to accuracy enhancement in normal specimens.